Development And Application Of Grape (Vitis Vinifera L.) EST-SNP Markers

Grape is the plant of Vitaceae Vitis and is one of the most important fruit trees inthe world. Because of the long history of cultivation and the large number of varieties,most of the grape varieties are difficult to distinguish using morphology characteristics,the different varieties using the same names or the same varieties using differentnames is a commom phenomenon, so we need a effective way to classify and identifyVitis plant. In this study, we utilize grape EST sequences from public databasesdevelop grape SNP locus, Establish detection system of CAPS marker and TSP marker,and analyzing the polymorphism information and dendrogram. The purpose of thispaper is to provide reference in grape genetic relationship identification, cultivaridentification molecular marker assisted breeding and other aspects. The main resultsare as follows:（1）42,493EST sequences were retrieved from different tissues of nine differentgrape genotypes in NCBI dbEST database and assembled into6,126contigs by CAP3software, then candidate SNP loci of the contigs were then screened by QualitySNPsoftware. The results showed that only1,195contigs contained candidate SNP loci. Atotal of5,032candidate SNPs were identified, and the amount of transversions,transitions and one base indel were1,800,2,896and336respectively, the averagefrequency of SNP occurred was at4.2SNP contig^-1. In order to improve the reliabilityof mined candidate SNP, the minor allele frequency was set at least30%in a candidateSNP locus, and the SNP flanking sequences would be perfect matching at least in5bp,to reduce false positive in small contig size and false negative in large contig size.（2） Establish a25μL CAPS marker reaction system: ddH₂O16.8μL,10×Buffer2.5μL, Mg²⁺1.5mmol·L^-1, forward and reverse primers are0.2μmol·L^-1respectively, dNTPs0.2mmol· L^-1, Taq DNA polymerase1U, template DNA50ng.The PCR program is: predenaturation at94℃for1min;35cycles containsdenaturation at94℃for30s, annealing at54℃for30s, extension at72℃for 1min;at last, extension at72℃for10min.（3） Establish a25μL TSP marker reaction system: ddH₂O15.6μL,10×Buffer2.5μL, Mg²⁺1.5mmol L^-1, locus-specific primer is0.2μmol L^-1, allele-specificprimer is0.96μmol L^-1, dNTPs0.2μmol L^-1, Taq DNA polymerase1U, templateDNA50ng. The PCR program is: predenaturation at95℃for10min,15cycles forlocus-specific primer and template DNA combination:94℃30s，58℃30s，72℃30s;5cycles for allele-specific primer and locus-specific primer competition:94℃10s，45℃30s;15cycles at last for allele-specific primer and template DNAcombination:94℃30s，53℃30s，72℃5s.（4） Designing28CAPS marker primers and22TSP markers primers anddetecting them using agarose gel electrophoresis in40grape materials,20CAPSmarker primers and10TSP markers primers have good results of agarose gelelectrophoresis bands and polymorphism. Genotyping statistics and Cluster analysisindicate that: all of the SNP locus contain2alleles, the average polymorphisminformation content（PIC） is0.336, the average Shannon’s information index is0.5105;most of the grape individuals from similar areas are clustered to a group, the resultsare reliable and can provide reference in grape genetic linkage map construction,molecular marker assisted breeding and other aspects.